¶Department of Genetics, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire 03755.

4

From the ‡Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire 03755; §Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Lebanon, New Hampshire 03756; arminja.n.kettenbach@dartmouth.edu.

Abstract

Cyclin-dependent kinase 1 (Cdk1) is an essential regulator of many mitotic processes including the reorganization of the cytoskeleton, chromosome segregation, and formation and separation of daughter cells. Deregulation of Cdk1 activity results in severe defects in these processes. Although the role of Cdk1 in mitosis is well established, only a limited number of Cdk1 substrates have been identified in mammalian cells. To increase our understanding of Cdk1-dependent phosphorylation pathways in mitosis, we conducted a quantitative phosphoproteomics analysis in mitotic HeLa cells using two small molecule inhibitors of Cdk1, Flavopiridol and RO-3306. In these analyses, we identified a total of 24,840 phosphopeptides on 4,273 proteins, of which 1,215 phosphopeptides on 551 proteins were significantly reduced by 2.5-fold or more upon Cdk1 inhibitor addition. Comparison of phosphopeptide quantification upon either inhibitor treatment revealed a high degree of correlation (R(2) value of 0.87) between the different datasets. Motif enrichment analysis of significantly regulated phosphopeptides revealed enrichment of canonical Cdk1 kinase motifs. Interestingly, the majority of proteins identified in this analysis contained two or more Cdk1 inhibitor-sensitive phosphorylation sites, were highly connected with other candidate Cdk1 substrates, were enriched at specific subcellular structures, or were part of protein complexes as identified by the CORUM database. Furthermore, candidate Cdk1 substrates were enriched in G2 and M phase-specific genes. Finally, we validated a subset of candidate Cdk1 substrates by in vitro kinase assays. Our findings provide a valuable resource for the cell signaling and mitosis research communities and greatly increase our knowledge of Cdk1 substrates and Cdk1-dependent signaling pathways.

Ratio distribution of phosphopeptides in Cdk1 and control treated mitotically-arrested HeLa cells.A and B, Frequency plots of log2 phosphopeptides ratios upon addition of Flavopiridol (A) or RO-3306 (B). C and D, Volcano plots of phosphopeptide ratios in Flavopiridol (C) or RO-3306 (D) treated mitotically-arrested HeLa cells. E, Correlation plot of log2 ratios of all phosphopeptides upon addition of Flavopiridol or RO-3306 compared with control. F, Correlation plot of log2 ratios of phosphopeptides quantified with a p value of less than 0.05 upon addition of Flavopiridol or RO-3306 compared with control.

Enrichment of candidate Cdk1 substrates in G2- and M-phase specific mRNA transcripts. Enrichment of candidate Cdk1 substrates identified by Flavopiridol or RO-3306 in cell cycle genes of different phases. Cell cycle genes in HeLa cells are sorted based on their peak expression during cell cycle progression. Each gene was assigned an angle range from 0° to 360° based on its peak time. Enrichment of substrates in cell cycle genes from each sliding window was calculated using Fischer's exact test over a sliding window of 30° with a 10° overlap between neighboring windows. Blue to yellow gradient indicated enrichment of candidate Cdk1 substrates in different cell cycle phases.

Validation of candidate Cdk1 substrates by in vitro kinase assay.A, Selected candidate Cdk1 substrates were expressed in bacteria, purified, and in vitro phosphorylated with Cdk1/cyclin B, Kinase reactions were quenched, separated by SDS-PAGE, trypsin digested and analyzed by LC-MS/MS. Spectra of phosphopeptides identified by in vitro kinase assay and in cells upon inhibitor treatment were aligned for comparison. B, Aligned, reciprocal spectra of Cdk1 inhibitor-sensitive phosphopeptides collected in vitro (top) and in cells (bottom).